Acetylcholine (ACh), a neurotransmitter candidate in the central nervous system, is stored in multiple locations with a cholinergic nerve terminal, the cytoplasm and the vesicles. The division of ACh into separate sites suggests that each site might have functional significance. However, the respective physiological roles or the precise interrelationship between the pools requires further elucidation. Some investigators have proposed that the cytoplasmic pool is the site of ACh formation and the vesicular pool is the site of ACh release. Conversely, others have suggested that the cytoplasm is the site of ACh formation and release. In both of these cases, the nerve ending in compartmentalized to provide only one mode of transmitter release. In contrast, the experiments described in this proposal will test the hypothesis that a central cholinergic nerve terminal is compartmentalized to provide multiple modes of transmitter release, that is, that ACh is not only stored in separate compartments but also that it is formed in and released from separate sites. Also, they will test whether the cytoplasmic pool of ACh is broken down during in vitro depolarization of mouse and rat brain tissue to provide choline and possibly acetate for the formation of ACh at the vesicular pool. Also, the experimental designs will test whether a soluble form of the synthetic enzyme ChAT forms. cytoplasmic ACh and whether a membrane associated form of ChAT, solubilized from cholinergic nerve endings with the detergent Triton DN 65, forms vesicular ACh. Also, it will be determined whether the soluble and membrane-bound forms of ChAT differ in hydrophobicity using Phenyl Sepharose chromatography. The significance of the proposed research will be the determination of why ACh is stored, synthesized and released from separate sites within the central cholinergic nerve ending; also the potential interrelationship between these separate storage, synthetic and release sites.